Bio-macromolecules, such as proteins, nucleic acids and polysaccharides, may often partially occur in the form of multimers, such as dimers, trimers or higher oligomers or aggregates. In, for example, recombinant DNA technology, where desired polypeptides or proteins are produced in host organisms and isolated from cell extracts under conditions and in concentrations quite different from those in their natural environment, the conditions may favour the formation of such multimers through intermolecular disulphide linkages or other covalent bonds, or through non-covalent interactions.
The presence of such multimers of a target macromolecule are many times undesired. For instance, if the macromolecule is intended for therapeutic use, the multimeric forms of the macromolecule may have lower or lack the biologic activity, or even cause undesired side-effects.
Prior art methods for analyzing protein or peptide multimers in a sample include, for example, size-exclusion chromatography and immunoassays. Whereas size-exclusion chromatography is relatively slow and complex to perform, the immunoassays require the use of specific monoclonal antibodies to the target proteins or peptides.
There is therefore a need for a method which can conveniently and rapidly analyse the presence of multimers of bio-macromolecules, for example in the purification of proteins or peptides, or when optimizing the conditions for protein purification.
In a solution of a biomolecule, such as a protein or peptide, not all biomolecules may have the expected activity, i.e. the ability to interact specifically with one particular ligand. For instance, a protein prepared by genetic engineering may have molecules incorrectly folded, or protein molecules may have been damaged during purification. Therefore, in a biological context it is the biologically active concentration that is relevant rather than the total concentration of the protein which merely indicates the amount of the biomolecule per unit volume.
Whereas the total concentration of e.g. a protein is typically measured by UV or NIR absorption spectrometry (which does not distinguish between active and inactive molecules), the active concentration of a biomolecule may conveniently be measured by biosensor technology, wherein a sample containing the biomolecule is contacted with a sensor surface with a specific ligand immobilized thereon, and the association/dissociation process at the surface is monitored.
In the determination of active concentration using biosensor technology, analyte concentrations can be determined without reference to a calibration standard. This method, which is usually referred to as Calibration-Free Concentration Analysis (CFCA), relies upon measurement of analyte binding to a target immobilized on a sensor surface at varying flow rates under conditions where the observed rate of binding is partially or completely limited by transport of analyte molecules to the sensor surface, i.e. partially or completely controlled by diffusion.
It is an object of the present invention to provide a method for analysing macromolecule multimers using such a sensor-based method for active concentration determination.